A long-term goal of my laboratory is to elucidate the molecular basis of signal transduction by Tumor necrosis factor (TNF) receptor associated factors (TRAFs), which are the major signal transducers of TNF receptors, IL-1 receptors, Toll-like receptors, and T-cell receptors. During the previous funding period, we have focused our effort on the molecular interactions of the TRAF C-terminal domain with receptors and other upstream signaling proteins. In this application, we propose the new initiative of elucidating the molecular mechanisms of TRAF downstream signaling, which centers on the activation of the TAK1 kinase complex composed of TAK1 and the adapter proteins TAB1 and TAB2. Two modes of TAK1 activation mechanism have been implicated. While TRAFs activate TAK1 via TAB2 in a Lys63-linked polyubiquitination pathway, XIAP activates TAK1 via TAB1 in an unknown ubiquitination-independent pathway. Our preliminary data support an emerging unifying principle of oligomerization in TAK1 activation by both TRAFs and XIAP. In this application, we propose a series of crystallographic, biochemical, biophysical and cell biological studies to address the molecular mechanisms of TAK1 activation and inhibition. The following specific aims are proposed. Aim 1. Role of TRAF trimerization and K63-linked ubiquitination in TAK1 activation Aim 2. Role of TRAF6 deubiquitination in terminating TAK1 activation: specificity of the deubiquitinase A20 for TRAF6 Aim 3. Role of dimerization in XIAP and TAB1 mediated TAK1 activation